Impact of iron availability on fecal
microcosms: modeling the effect of diet on
microbial symbiotic populations
Kelli L. Palmer
MBL Microbial Diversity 2008
Abstract:
The human body plays host to a diverse community of microbial
symbionts. The intestinal symbiotic community in particular is critical
for bodily function, shaping immune system development in infancy
and nutrient adsorption processes throughout life. Both the human
body and its symbionts require iron, a necessary cofactor for many
metabolic processes. It is likely that the main source of iron for
intestinal symbionts is excess dietary iron that passes through the
intestine during digestion. Thus, dietary iron intake has the potential
to significantly impact microbial community structure in the intestine.
In this study, the hypothesis that available iron impacts intestinal
community structure was tested. Anaerobic fecal microcosms were
used to model the intestinal environment, and varying iron or chelator
treatments were used to model host dietary changes. Restriction
fragment length polymorphism (RFLP) profiles of amplified bacterial
16S rDNA genes indicate that iron availability does not have a
significant impact on symbiotic community structure. These results
suggest that the intestinal microbial community is resilient in the face
of marked dietary fluctuations.
Introduction
Iron is a required nutrient for almost every form of life on the planet. Many
metabolic enzymes (metalloenzymes) require iron as a cofactor, thus iron
availability can govern an organism’s metabolic potential. Conversely, and
perhaps paradoxically, excess iron is exceedingly toxic to aerobic organisms, as
iron and molecular oxygen can interact to form highly reactive oxygen radicals
capable of damaging nucleic acids and other cellular constituents 1. For these
reasons, iron uptake and cellular iron pools are tightly regulated in aerobic
organisms. This includes the human body, where proteins such as transferrin
and lactoferrin circulate throughout the body, tightly binding excess iron where
appropriate and distributing it where needed. Interestingly, it is thought that one
of the primary innate immune defenses of the human body is iron sequestration
by proteins such as transferrin, as most invading pathogens will require iron for
rapid growth during infection 2.
How does the human body regulate iron levels in order to simultaneously provide
enough for cellular processes as well as prevent potential toxic iron effects and
cheating by invading pathogens? Iron is lost continually by the body through
sloughing of dead cells (0.5-2 mg per day), thus new iron must be input into the
system. This occurs through the diet. In fact, iron levels in the human body are
regulated solely at the level of dietary intake, as there is no active mechanism for
iron efflux. Cells lining the small intestine take up iron from dietary constituents
by enzymatically reducing ferric iron (thought to be the major form of iron in
foods) to ferrous iron, which can then be transported inside the cell. Other
mechanisms of iron uptake, such as heme transport, have also been
documented. Excess iron in the diet remains in the small intestine and passes
through the remainder of the gastrointestinal tract to be excreted with other fecal
matter 3. The amount of dietary iron intake and fecal iron excretion in healthy
individuals is correlated 4.
While tight regulation of iron uptake and cellular iron pools can help the human
body fight infections by pathogenic microbes, other microbes may be significantly
affected by these efforts: the human microbiome. We now know that the human
body plays host to a diverse community of microbes that shape development and
critical bodily functions. Arguably one of the most important and well studied of
these is the intestinal community, where ~1014 microbes can reside
5
.
Maintenance of the intestinal microbiome may be critical for health, as evidence
mounts that this community can shape immune system development 6 as well as
prevent inflammatory bowel diseases 7 and promote obesity 8.
What are the potential sources of iron for the intestinal community? The major
source of iron is likely to be excess iron that is not absorbed from the diet—the
‘leftovers.’ Thus, fluxes in dietary iron intake have the potential to significantly
impact intestinal microbiome structure. In this study, the hypothesis that dietary
iron levels impact intestinal microbial community structure was tested.
Materials and Methods
Anaerobic microcosms. Anaerobic media were prepared as follows. For
freshwater base and vitamin mix components, see Microbial Diversity 2008
laboratory manual. Base medium (1 mL 0.1% rezasurin; 10 mL 100X freshwater
base; 1 mL 1000X vitamin mix; 1 mL 1000X vitamin B12 mix; 20 mL 1 M MOPS,
pH 7.2; 5 mL 1 M NH4Cl; 5 g tryptone; 5.88 g NaHCO3; 940 mL double distilled
water) was boiled in a round-bottom flask under a constant flow of 80:20 N2:CO2
gas for 15 min. The mixture was allowed to cool under gas flow, and then tightly
capped. In the anaerobic chamber, 48.5 mg cysteine, 2 mL 0.2 M Na2S, and 1
mL 1 M potassium phosphate buffer, pH 6.8, was added. After incubation with
occasional mixing for 30 min, 30 mL media were dispensed into anaerobic
bottles, sealed, and autoclaved for 45 min. After autoclaving, 0.6 mL of a 0.5 M
anaerobic glucose stock was added to all bottles. For some bottles, 0.3 mL of a
10.37 mM anaerobic ferrous sulfate stock, 0.3 mL of a 10.37 mM anaerobic ferric
citrate stock, or 0.5 mL of an 18.27 mM anaerobic deferoxamine stock was
added. Sterile, anaerobic double distilled water was used to balance volumes in
all bottles. Microcosms were inoculated under a flow of 80:20 N2:CO2 gas with
fresh fecal material (~100 !L) collected clean-catch from a healthy volunteer and
were incubated at 37"C without shaking. Microcosms were performed in triplicate
for each treatment.
RFLP. At 8 h, 48 h, and 7 days post-inoculation, approximately 1 mL sample was
removed from microcosms and stored at -80"C prior to analysis. Total genomic
DNA was isolated from duplicate microcosm samples (for a total of 8 samples
per timepoint) using the PowerSoil DNA isolation kit (MoBio Labs). Samples were
eluted in sterile, double distilled water, and 2 !L each were subsequently used in
a standard 25 !L PCR reaction using 15 pmol each 8F and 1492R universal
eubacterial primers and 2X master mix (Promega). PCR reactions were
denatured at 95"C for 5 min, cycled [95"C, 30 s; 46"C, 30 s; 72"C, 90 s] 30
times, and extended at 72"C for 5 min. Genomic DNA yields were roughly
equivalent for all samples (data not shown). PCR products were confirmed by
agarose gel electrophoresis and purified using a QiaQuick PCR purification kit
(Qiagen). For each sample, 500 ng PCR product was used in a 30 !L final
reaction volume with 1 !L MspI (New England Biolabs). Restriction digests were
incubated at 37"C for 1-3 h and were analyzed by electrophoresis on 1%
agarose gels.
Ferrozine assay. At 7 days post-inoculation, ~250 !L from each microcosm was
sampled and immediately diluted 1:1 in an equal volume 0.5 N HCl. Diluted,
acidified samples were centrifuged at 10,000 x g for 10 minutes to pellet cell
debris, and 20 !L supernatant from each sample was used for analysis by the
ferrozine assay as follows. Ferrozine assay buffer (1 g/L ferrozine in 49 mM
HEPES buffer, pH 7.0) was brought to room temperature and 980 !L were
dispensed into Eppendorf tubes. Twenty !L standard (0.05, 0.1, 0.2, 0.4 or 0.8
mM acidified ferrous ethylene diammonium sulfate) or test sample was added,
and the reaction was allowed to proceed at room temperature for 5 min before
absorbance was measured at 562 nm.
Results
Modeling dietary changes in the intestinal environment. To test the
hypothesis that dietary iron fluxes impact intestinal microbial community
structure, an appropriate intestinal model was required. For this purpose I chose
anaerobic fecal microcosms. The medium used for the fecal microcosms
consisted of a buffered salts base with 0.5% tryptone (amino acids) and 10 mM
glucose added as carbon sources. Vitamins were also added to supplement
growth. For control microcosms, no iron was added. Three experimental
treatments were used to mimic various dietary changes: 100 !M ferrous sulfate,
100 !M ferric citrate, and 300 !M deferoxamine (an iron chelator).
RFLP profiles of fecal microcosms. After 8 h incubation at 37"C, all fecal
microcosms, independent of treatment, were turbid. RFLP profiles of 8 h
microcosm communities showed no obvious differences between treatments
(Figure 1).
After 48 h incubation, RFLP profiles from all treatments showed additional bands
between 510 and 1020 base pair DNA markers (Figure 2), indicating that a
subpopulation of the initial inoculum had grown to detection by this method. No
striking differences between treatments were observed although abundance of
bands near the 300 base pair marker in ferric citrate-treated microcosms
appeared to deviate from that of other treatments.
After 7 d incubation, RFLP profiles of ferric citrate-treated microcosms indicated
that one or more bacterial species within in the microcosms had decreased in
abundance relative to other treatments, while one or more bacterial species
increased in abundance (Figure 3). These results suggest that the presence of
ferric citrate influenced fecal microcosm community structure. Microscopy of all
fecal microcosms revealed a diversity of cell types, including short and long nonmotile rods, motile rods, and cocci (data not shown). No cell morphologies
dominated fecal microcosms with different treatments, and no obvious
differences were observed microscopically amongst different treatments.
Iron levels in fecal microcosms. To confirm that varying iron levels were
present in fecal microcosms, the ferrozine assay was used to measure iron levels
in these cultures. Ferrozine forms a stable complex with ferrous iron, producing a
magenta color that can be measured spectrophotometrically. Samples from 7 d
microcosms were used in the ferrozine assay. Results are summarized in Table
1. Unfortunately, only samples from ferrous sulfate treated microcosms fell within
the standard curve generated. However, some data can be inferred, including the
relative abundance of iron in the ferric citrate treated microcosms compared to
the control and deferoxamine treated microcosms.
Discussion
In this study, fecal microcosms were used to model the human intestine, and
varying microcosm treatments were used to mimic dietary flux in iron intake. I
chose to use ferrous sulfate and ferric citrate to mimic dietary iron, as these iron
complexes are commonly used as dietary supplements and can be found in
foodstuffs such as fortified cereals. It is important to note that citrate is a carbon
source that could potentially affect community structure; however, the
concentration used in this study (100 !M) was low and was thus unlikely to
significantly impact ferric citrate treated communities. The iron chelator
deferoxamine was also chosen as a treatment as this chelator is commonly used
in clinical therapies to treat iron overload syndromes such as haemochromatosis.
Chelation of dietary or bodily iron could also impact intestinal community
structure.
It is clear from 8 h RFLP data that the intestinal population that grew rapidly in
fresh fecal microcosms was impervious to the amount of iron available. First,
there appeared to be no effect of excess iron (ferrous or ferric) on microcosm
community structure. Addition of the chelator deferoxamine, intended to create
an iron-limited environment, also had no effect. It is possible that the
concentration of deferoxamine used in this study (300 !M), used in a previous
study to inhibit aerobic growth of several bacterial species 9, is not sufficient to
create an anaerobic iron-limited environment. It is also important to note that a
RFLP profile was not performed on the original fecal inoculum, which would have
given some information on the structure of the original fecal community.
Comparison of this RFLP with the 8 h RFLP data would have indicated the extent
of enrichment imposed on the community by my experimental conditions.
Data from 48 h and especially 7 d RFLP profiles show that there is some effect of
ferric citrate on fecal microcosm community structure. I question the relevance of
the 7 d data as this period of incubation does not accurately reflect retention
times of fecal material in the intestine. In addition, high performance liquid
chromatography analysis of culture supernatants from 7 d microcosms indicated
that microcosm fermentation products were uniform across treatments (data not
shown).
Collectively, these data suggest that there is little effect of dietary iron on
intestinal
communities.
Interestingly,
methane
was
produced
by
fecal
microcosms (data not shown). In the future, it would be interesting to see the
effect of varying iron on archaeal populations in the intestine, as methanogenic
archaea are known for their remarkably high number of iron-sulfur clusters
10
. In
addition, given the stability of the in vitro fecal microcosms, it would also be
interesting to spike in ‘outsiders,’ like Escherichia coli O157 or Vibrio cholerae
and examine their ability to compete with the resident community for iron.
However, if these studies are to be pursued in the future, it will be important to
sample additional individuals for fecal microcosm inocula as well as include
RFLP analyses of initial inocula.
Acknowledgements
All the class and TAs. You are awesome!! And thanks to Bill and Tom—I suspect
the MBL won’t be the same without you.
References
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Figure 1.
1
2
3
4
1640
1020
510
300
Figure 1. RFLP profile of 8 h fecal microcosm samples. Shown are MspI
digestion patterns for bacterial 16S rDNA amplified from control (1), ferrous
sulfate (2), ferric citrate (3) and deferoxamine (4) treated fecal microcosms. Only
one replicate is shown for ferric citrate microcosms. Numbers on the left indicate
DNA ladder reference sizes in base pairs.
Figure 2.
1
2
3
4
1640
1020
510
300
Figure 2. RFLP profile of 48 h fecal microcosm samples. Shown are MspI
digestion patterns for bacterial 16S rDNA amplified from control (1), ferrous
sulfate (2), ferric citrate (3) and deferoxamine (4) treated fecal microcosms.
Numbers on the left indicate DNA ladder reference sizes in base pairs.
Figure 3.
1
2
3
4
1640
1020
510
300
Figure 3. RFLP profile of 7 d fecal microcosm samples. Shown are MspI
digestion patterns for bacterial 16S rDNA amplified from control (1), ferrous
sulfate (2), ferric citrate (3) and deferoxamine (4) treated fecal microcosms.
Numbers on the left indicate DNA ladder reference sizes in base pairs.
Table 1.
Sample
OD562nm
Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
0.03
0.065
0.109
0.222
0.447
Control-1
Control-2
Control-3
Fe (II)-1
Fe (II)-2
Fe (II)-3
Fe (III)-1
Fe (III)-2
Fe (III)-3
Deferox-1
Deferox-2
Deferox-3
0.004
0.009
0.008
0.033
0.033
0.03
0.024
0.024
0.022
0.008
0.007
0.007
[Fe] (!M)
50
100
200
400
800
~13 !M
~100 !M
~75 !M
~13 !M
Table 1. Iron levels in 7 d fecal microcosms. Iron levels were measured in
fecal microcosms by the ferrozine assay (see methods). Values for ferrous iron
standards are shown and amounts of ferrous iron in microcosms were calculated
from the standard curve generated (y = 0.5526x + 0.033; R2 = 0.9994). Samples
within the standard curve are highlighted in bold, italic text. Ferrozine assay
buffer alone was used as a blank.